Purification of heparinase I, II, and III from Flavobacterium heparinum

ABSTRACT

A purified heparinase I, II and III free of lyase activity and each having a molecular weight of 42,800 84,100, 70,800, respectively, are produced by culturing Flavobacterium heparinum. The kinetic properties of the heparinases have been determined as well as the conditions to optimize their activity and stability.

BACKGROUND OF THE INVENTION

The U.S. government has certain rights in the invention since thisinvention was made with government support under contract numberNIH-2R01-GM25810 awarded by the National Institutes of Health.

This invention generally relates to the purification andcharacterization of heparinase I, II, and III from Flavobacteriumheparinum and antibodies thereto.

Heparin and heparan sulfate represent a class of glycosaminoglycanscharacterized by a linear polysaccharide of D-glucosamine (1→4) linkedto hexuronic acid (Linhardt, R. J. (1991) Chem. Ind. 2, 45-50; Casu, B.(1985) Adv. Carbohydr. Chem. Biochem. 43, 51-134). Heparin and heparansulfate are complex carbohydrates that play an important functional rolein the extracellular matrix of mammals. These polysaccharides modulateand regulate tissue level events that take place either duringdevelopment under normal situations or wound healing and tumormetastasis under pathological conditions.

Much of the current understanding of heparin and heparan sulfatesequence has relied on studies of their biosynthesis (Linhardt, R. J.,Wang, H. M., Loganathan, D., and Bae, J. H. (1992) Biol. Chem. 267,2380-2387; Lindahl, U., Feingold, D., and Roden, L. (1986) TrendsBiochem. Sci. 11, 221-225; Jacobson, I., and Lindahl U. (1980) J. Biol.Chem. 255, 5094-5100; Lindahl, U., and Kjellen, L. (1987) in The Biologyof Extracellular Matrix Proteoglycans (Wight, T. N., and Mecham R., eds)pp. 59-104, Academic Press, New York). Recent efforts (Linhardt, R. J.,Rice, K. G., Kim, Y. S., Lohse, D. L., Wang, H. M., and Loganathan, D.(1988) Biochem. J. 254, 781-787; Linhardt, R. J., Turnbull, J. E., Wang,H. M., Loganathan, D., and Gallagher, J. T. (1990) Biochemistry 29,2611-2617) have focused on the application of enzymatic methods todepolymerize these complex polysaccharides into oligosaccharides thatcould then be structurally characterized (Linhardt, et al. (1992) Biol.Chem. 267, 2380- 2387; Linhardt, et al., (1988) Biochem. J. 254, 781-787; Loganathan, D., Wang, H. M., Mallis, L. M., and Linhardt, R. J.(1990) Biochemistry 29, 4362-4368).

Enzymatic methods for heparin and heparan sulfate depolymerization arevery specific and require mild conditions giving oligosaccharideproducts that closely resemble the glycosaminoglycans from which theywere derived. Two types of enzymes that degrade heparin and heparansulfate glycosaminoglycans are the polysaccharide lyases fromprokaryotic sources that act through an eliminative mechanism (Linhardt,R. J., Galliher, P. M., and Cooney, C. L. (1986) Appl. Biochem Biotech.12, 135-176), and the glucuronidases (hydrolases) from eukaryoticsources that act through a hydrolytic mechanism.

Prokaryote degradation of heparin and heparan sulfate has primarily beenstudied using enzymes derived from Flavobacterium heparinum (Linker, A.,and Hovingh, P. (1965) J. Biol. Chem. 240, 3724-3728; Linker, A., andHovingh, P. (1970) J. Biol Chem. 245, 6170-6175); Dietrich, C. P.,Silva, M. E., and Michelacci, Y.M. (1973) J. Biol. Chem. 248, 6408-6415;Silva, M. E., Dietrich, C. P., and Nader, H. B. (1976) Biochem. Biophys.Acta 437, 129-141). This bacterial degradation begins with the action ofthree (or possibly more) eliminases. These heparin lyases produceoligosaccharides with Δ₄,5 -unsaturated uronic acid residues at theirnon-reducing termini. These eliminases probably act in concert toconvert heparin and heparan sulfate to disaccharides.

Heparin lyases are a general class of enzymes that are capable ofspecifically cleaving the major glycosidic linkages in heparin andheparan sulfate. Three heparin lyases have been identified inFlavobacterium heparinum, a heparin-utilizing organism that alsoproduces exoglycuronidases, sulfoesterases, and sulfamidases thatfurther act on the lyase-generated oligosaccharide products (Yang, V.C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985)J. Biol. Chem. 260, 1849-1857; Galliher, P. M., Linhardt, R. J., Conway,L. J., Langer, R., and Cooney, C. L. (1982) Eur. J. Appl. Microbiol.Biotechnol. 15, 252-257). These lyases are designated as heparin lyase I(heparinase, EC 4.2.2.7), heparin lyase II (heparinase II, no EC number)and heparin lyase III (heparitinase EC 4.2.2.8). Although thespecificities of these enzymes are not completely known, studies usingpartially purified enzymes with heparin, heparan sulfate, andstructurally characterized heparin oligosaccharides have led to anunderstanding of the linkages susceptible to enzymatic cleavage(Lindhardt, et al., (1990), Lohse (1992), Rice, K. G., and Linhardt, R.J. (1989) Carbohydr. Res. 190, 219-233). The three purified heparinlyases differ in their capacity to cleave heparin and heparan sulfate:Heparin lyase I primarily cleaves heparin, heparin lyase IIIspecifically cleaves heparan sulfate and heparin lyase II acts equallyon both heparin and heparan sulfate (Linhardt, et al., 1986; Linhardt,et al., 1990).

Several Bacteroides sp. (Saylers, A. A., Vercellotti, J. R., West,S.E.H., and Wilkins, T. D. (1977) Appl. Environ. Microbiol. 33, 319-322;Nakamura, T., Shibata, Y., and Fujimura, S. (1988) J. Clin. Microbiol.26, 1070-1071) also produce heparinases, however, these enzymes are notwell characterized. A heparinase has also been purified to apparenthomogeneity from an unidentified soil bacterium (Bohmer, L. H., Pitout,M. J., Steyn, P. L., and Visser, L. (1990) J. Biol. Chem. 265,13609-13617). This enzyme differs from those isolated fromFlavobacterium heparinum in its molecular weight (94,000), pI (9.2),amino acid composition and kinetic properties (K_(m) of 3.4 μM andV_(max) of 36.8 μmol/min, pH optimum of 7.6).

Three other heparin lyases, partially purified from Flavobacterium sp.Hp206, have molecular weights of 64,000, 100,000, and 72,000, asreported by Yoshida, K., Miyazono, H., Tawada, A., Kikuchi, H.,Morikawa, K., and Tokuyasu, K. (1989) 10th Annual Symposium ofGlycoconjugates, Jerusalem, different from heparin lyases I-III.

The heparin lyases of F. heparinum are the most widely used and beststudied (Lindhardt, (1986)). Linker and Hovingh (1970) first separatedthese lyase activities, fractionating a crude lyase fraction into aheparinase (heparin lyase I) and a heparitinase (heparin lyase III).Both activities were purified by 50-100-fold, but no physicalcharacterization of these enzymes was performed.

Dietrich and co-workers (Dietrich, et al., 1973); Silva, et al., (1976);Silva, M. E., and Dietrich, C. P. (1974) Biochem. Biophys. Res. Commun.56, 965-972; Michelacci, Y. M., and Dietrich, C. P. (1974) Biochem.Biophys. Res. Commun. 56, 973-980) and Ototani and Yosizawa (Ototani,N., and Yosizawa, Z. (1978) J. Biochem. (tokyo) 84, 1005-1008; Ototani,N., and Yosizawa, Z. (1979) Carbohydr. Res. 70, 295-306; Ototani, N.,Kikiuchi, M., and Yosizawa, Z. (1981) Carbohydr. Res. 88, 291-303;Ototani, N., and Yosizawa, Z. (1981) Proceedings of the 6thInternational Symposium on Glycoconjugates, pp. 411-412, September20-25, Tokyo, Japan Scientific Press, Tokyo) isolated three lyases, aheparinase (heparin lyase I) and two heparitinases, from F. heparinum.The heparinase acted on heparin to produce mainly trisulfateddisaccharides (Dietrich, C. P., and Nader, H. B. (1974) Biochem.Biophys. Acta 343, 34-44; Dietrich, C. P., Nader, H. B., Britto, L. R.,and Silva, M. E. (1971) Biochem. Biophys. Acta 237, 430-441); Nader, H.B., Porcionatto, M. A., Tersariol, I. L. S., Pinhal, M. S., Oliveira, F.W., Moraes, C. T., and Dietrich, C. P. (1990) J. Biol. Chem.265,16807-16813) purified two heparitinases (called heparitinase I andII, possibly corresponding to heparin lyases II and III, although nophysical properties of these enzymes were presented) and characterizedtheir substrate specificity toward heparin and heparan sulfate.Heparitinase I degraded both N-acetylated and N-sulfated heparan sulfatewhile heparitinase II degraded primarily N-sulfated heparan sulfate.

McLean and Co-workers described the specificity of a partially purifiedheparinase II (Moffat, C. F., McLean, M. W., Long, W. F., andWilliamson, F. B. (1991) Eur. J. Biochem. 197, 449-459; McLean, M. W.,Long, W. F., and Williamson, F. B. (1985) in Proceedings of the 8thInternational Symposium on Glycoconjugates, pp. 73-74, September,Houston, Paeger Publishers, New York; McLean, M. W., Bruce, J. S., Long,W. F., and Williamson, F. B. (1954) Eur. J. Biochem. 145, 607-615).Although no evidence of homogeneity or any physical properties forheparinase II were presented, the broad specificity on various polymericsubstrates (Moffat, et al., (1991)) identifies the enzyme as heparinlyase II (Lindhardt, et al., (1990); McLean, et al., (1985).

Linhardt et al. (1984) Appl. Biochem. Biotech. 9, 41-55) reported thepurification of heparinase (heparin lyase I) to a single band onSDS-PAGE. Affinity purification of heparin lyase I on heparin-Sepharosefailed, apparently due to degradation of the column matrix. Sufficientquantities of pure heparin lyase I for detailed characterization studiesand amino acid analysis were first prepared by Yang et al. (1985).Heparin lyase I was used to prepare polyclonal antibodies in rabbits foraffinity purification of heparin lyase I, but excessively harshconditions required to elute the enzyme resulted in substantial loss ofactivity (Lindhardt, (1985)). Yang, V. C., Bernstein, H., Cooney, C. L.,and Langer, R. (1987) Appl. Biochem. Biotech. 16, 35-50)) also describeda method to prepare heparin lyase I.

Seikagaku Co. has recently orally reported the molecular weights oftheir commercial enzymes corresponding to heparin lyase I-III to be43,000, 84,000, and 70,000, respectively (Yoshida, K. (1991)International Symposium on Heparin and Related Polysaccharides,September 1-6, Uppsala, Sweden). These reports are in close agreement tothe molecular weights described herein, but no details of theirpurification or characterization methods have been published.

Heparin lyases have been used to establish the presence of heparin inmixtures of proteoglycans (Kanwar, Y. S., and Farquhar, M. G. (1979)Presence of heparan sulfate in the glomerular basement membrane. Proc.Natl. Acad. Sci., USA 76, 1303-1307), to depolymerize heparin andheparan sulfate to characterize the structure of the resultingoligosaccharides (Linhardt, R. J., Loganathan, D. Al-Hakim, A., Wang,H.-M., Walenga, J. M., Hoppensteadt, D., and Fareed, J. (1990)Oligosaccharide mapping of low molecular weight heparins: structure andactivity differences. J. Med. Chem. 33, 1639-1645; Linhardt, R. J.,Rice, K. G., Kim, Y. S., Lohse, D. L., Wang, H. M., and Loganathan, D.(1988). Mapping and quantification of the major oligosaccharidecomponents of heparin. Biochem. J. 254, 781-787; Merchant, Z. M., Kim,Y. S., Rice, K. G., and Linhardt, R.J. (1985). Structure ofheparin-derived tetrasaccharides. Biochem. J. 229, 369-377; Turnbull, J.E., and Gallagher, J. T. (1988) Oligosaccharide mapping of heparansulphate by polyacrylamide-gradient-gel electrophoresis andelectrotransfer to nylon membrane. Biochem J. 251, 597-608), to producelow molecular weight heparin preparations with anticoagulant andcomplement inhibitory activities (Linhardt, R. J., Grant, A., Cooney, C.L., and Langer, R. (1982) Differential anticoagulant activity of heparinfragments prepared using microbial heparinase. J. Biol. Chem. 257,7310-7313; Linhardt, R. J., and Loganathan, D. (1990a). Heparin,heparinoids and heparin oligosaccharides: structure and biologicalactivity. In C. G. Gebelein (Ed.), Biomimetic Polymers (pp. 135-173).New York: Plenum Press; Sharath, M. D., Merchant, Z. M., Kim, Y. S.,Rice, K. G., Linhardt, R. J., and Weiler, J. M. (1985) Small heparinfragments regulate the amplification pathway of complement.Immunopharmacology 9, 73-80) and to remove heparin from the circulation(Langer, et al., 1982). Heparin depolymerising enzymes are excellenttools to understand the role of heparin-like molecules in theextracellular matrix or to be used in different tissue microenvironmentsto modulate and alter the extracellular matrix in a highly specificmanner. However, studies utilizing heparin lyases are hampered bydifficulties in purifying the enzymes from Flavobacterium heparinum,especially with regard to separation of the three enzymes from eachother (Linhardt, et al., 1985). Specifically, the capacity of heparinlyase II to cleave both heparin and heparan sulfate makes it difficultto distinguish from heparin lyase I which cleaves heparin and heparinlyase III which cleaves heparan sulfate.

Although all three of these heparin/heparan sulfate lyases are widelyused, with the exception of heparin lyase I, there is no information onthe purity or physical and kinetic characteristics of heparinase II andheparinase III. The absence of pure heparin lyases, resulting inambiguities with respect to substrate specificity. This is due tocontamination of other lyases in the preparation, and a lack ofunderstanding of the optimal catalytic conditions and substratespecificity has stood in the way of the use of these enzymes as reagentsfor the specific depolymerization of heparin and heparan sulfate intooligosaccharides for structure and activity studies, and for use inclinical studies.

It is therefore an object of the present invention to provide a methodfor purification and characterization of heparinase I, heparinase II,and heparinase III.

It is a further object of the present invention to provide purified andcharacterized heparinase I, heparinase II, and heparinase III.

It is a still further object of the present invention to provide theconditions for optimal use and peptide map of the purified heparinase IIand heparinase III.

It is another object of the present invention to provide the amino acidcompositions of the three heparinases.

It is another object of the present invention to provide antibodies forheparinase I, II, and III which can be used in the purification andcharacterization of heparinases.

SUMMARY OF THE INVENTION

A single, reproducible scheme to simultaneously purify all three of theheparin lyases from F. heparinum to apparent homogeneity and free ofcontaminating lyases is disclosed herein. Heparin lyase I (heparinase,EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III(heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecylsulfate-polyacrylamide gel electrophoresis) and isoelectric points (byisoelectric focusing) of M_(r) 42,800, pI 9.1-9.2, M_(r) 70,800, pI9.9-10.1, respectively. Their amino acid analyses and peptide mapsdemonstrate that while these proteins are different gene products theyare closely related. The kinetic properties of the heparin lyases havebeen determined as well as the conditions to optimize their activity andstability.

The purification and characterization of heparinase II fromFlavobacterium heparinum is described. The Michelis-Menton constantsare: Heparin lyase II (with heparin), V(max)=15.04, K_(m) =9.23 μM(0.129 mg/ml); Heparin lyase II (with heparan sulfate), V(max)=46.95,K_(m) =43.43 μM (0.869 mg/ml). The approximate pI of the lyasecalculated from agarose IEF using a pH gradient from 9-11 is around 8.9.The optimum temperature for heparin lyase II (both heparin and heparansulfate) is 35° C. The activity is greater at higher temperatures butthe stability is greatly reduced. The optimum pH for activity for thelyase: (with heparin), pH=7.3 and (with heparan sulfate), pH=6.9.

The purification and characterization of heparinase III (EC 4.2.2.8)from Flavobacterium heparinum is described. The Michelis-Mentonconstants are V(max)=277.01, K_(m) =109.97 μM (0.780 mg/ml). Theapproximate pI of the lyase was calculated from agarose IEF using a pHgradient from 9-11 and was found to be 9.2. The optimum temperatures forthe heparin lyase III activity is 35° C. The activity is higher athigher temperatures for the enzyme but the stability is greatly reduced.The optimum pHs for heparin lyase III is pH=7.6. The substratespecificity of heparinase III is for the hexosamine-glucuronic acidlinkages of the heparan sulfate backbone. The enzyme is a monomericprotein, very different from heparinase I and II in size and activity.It is possible to use heparinase III to release heparin-like chains inthe extracellular matrix, for both sequencing and eliciting heparinbased cellular response.

Salt effects were not observed for either heparinase II or heparinaseIII. Four different salts were used to confirm that salt effects and notion effects were tested.

Methods for the preparation and use of monoclonal antibodies to thethree heparinases are also described. The antibodies are useful forisolation, detection and characterization of the heparinases,individually and as a group, and in studies involving substratespecificity, enzyme inhibition and active site mapping.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the HA-HPLC fractionation of heparin lyases. Theprotein (A₂₈₀) is the solid line. The activity (unit/ml) toward heparin(solid circles) and activity (unit/ml) toward heparan sulfate (solidsquares) are shown with cross-hatching to indicate the portion of thepeaks that were collected.

FIG. 2 is Mono-S FPLC fractionation of heparin lyases: a, heparin lyaseI, and b, heparin lyase III. The arrow indicates the start of the saltgradient elution, and the cross-hatching indicates the portion of thepeaks that were collected.

FIG. 3 is a GPC-HPLC fractionation of heparin lyases. a, heparin lyaseI; b, heparin lyase II; c, heparin lyase III; and d, molecular weightstandards (M_(r)) consisting of thyroglobulin (bovine, 670,000), gammaglobulin (158,000), ovalbumin (44,000), myoglobin (horse, 17,000), andcyanocobalamin (1350). The cross-hatching indicates the portion of thepeaks that were collected.

FIG. 4 is an SDS-PAGE in a 12% discontinuous polyacrylamide gel underreducing conditions. Two μg each of heparin lyase I (lane a), heparinlyase II (lane b), heparin lyase III (lane c), and molecular weightstandards (lane d). Shown to the right are the mass of the molecularweight standards in kDa.

FIG. 5. Panel A: Western Blot of SDS-PAGE gel using M2-A9. (a) heparinlyase I; (b) heparin lyase II; (c) heparin lyase III; (d) Flavobacteriumheparinum cell homogenate. Arrows indicate bands of interest. Thisanalysis demonstrates the ability of this MAb to detect the presence ofheparin lyases that are either purified or present in homogenizedcellular material.

Panel B: SDS-PAGE analysis of purified heparin lyases. (a) heparin lyaseI; (b) heparin lyase II; (c) heparin lyase III; (d) molecular weightmarkers. Arrows indicate bands of interest.

FIG. 6 is a map of the tryptic digest of heparinase II and III. Panel Ais heparinase II and Panel B is heparinase III.by

DETAILED DESCRIPTION OF THE INVENTION

I. Purification and Characterization of Heparinase I, II, and III.

A single, reproducible scheme to simultaneously purify all three of theheparin lyases from F. heparinum to apparent homogeneity is describedherein.

EXPERIMENTAL PROCEDURES

Materials

Enzyme assays and absorbance measurements were done on a UV 160spectrophotometer from Shimadzu connected to a Fisher Scientific Isotampmodel 9100 refrigerated circulating water bath. Fermentations wereperformed in a two-liter stirred tank fermenter from Applikon.Centrifugation was done on a Sorval RC-5 refrigerated centrifuge in aGSA rotor from Du Pont. HPLC was performed using a LDC Milton-RoyConstametric IIIG pump, a Rheodyne 7125 injector, a Jule Linear GradientFormer, and an ISCO model UA-5 absorbance monitor with a 280-nm filter.The hydroxylapatite HPLC column 1×30 cm connected in series with a 1×5cm guard column was from Regis, the Mono-S FPLC column was fromPharmacia LKB Biotechnology Inc., the C₁₈ column was from Vydac, and theBio-Sil gel permeation HPLC column was from Bio-Rad. The capillary zoneelectrophoresis system and the silica capillaries were from Dionex. TheMini-Protein II electrophoresis chamber, a model 1405 horizontalelectrophoresis cell, and a model 1420B power source were from Bio-Rad.The tube gel electrophoresis equipment was from E-C Apparatus Corp. Theprecast agarose IEF gels were from Iso-labs, and the prestainedmolecular weight markers and the Rapid Coomassie™ stain were fromDiversified Biotech. The Bio-Gel HT hydroxylapatite was from Bio-Rad andthe QAE-Sephadex was from Sigma. Pressure filtration units and 25- and43-mm PM-10 filters were from Amicon. Heparin (porcine mucosal sodiumsalt) was from Celsus, heparan sulfate, dermatan sulfate, andchondroitin sulfate A, C, D, and E were from Seikagaku. Bovine serumalbumin, lactose, protamine (free base), bromphenol blue, naphthol red,cytochrome c (bovine heart type VA), hyaluronic acid, CAPS, bis-Tris,HEPES, TES, dithiothreitol, MOPS, mercaptoethanol, iodoacetamide, andtrypsin were for Sigma. The Coomassie reagent for the protein assay wasfrom Bio-Rad. All water used in reagents was deionized and distilled inglass.

Assays.

The spectrophotometer was adjusted to the optimum temperature of theparticular lyase being assayed. A 700 μl quartz microcuvette containing400 μg of substrate in 50 mM sodium phosphate buffer (containing 100 mMsodium chloride for heparin lyase I) was thermally equilibrated. Ameasured quantity of lyase was added, bringing the final volume to 400μl and the cuvette was gently mixed. The microcuvette was thenimmediately returned to the spectrophotometer and the change ofabsorbance at 232 nm was measured at 10 seconds intervals over 3 min.The activity was measured from the change of absorbance/unit time usingan extinction coefficient of 3800 M⁻¹ for products. The specificactivity was then calculated by dividing the micromoles of productproduced per minute by the milligrams of protein in the cuvette. Themolecular weights used for heparin, heparan sulfate, and the chondroitinsulfates were 14,000, 20,000 and 25,000, respectively, Rice, K. G., andLinhardt, R. J. (1989) Carbohydr. Res. 90, 219-233. Proteinconcentration was measured by the Bradford assay, Bradford, M. M. (1976)Anal. Biochem. 72, 248-254, based on a bovine serum albumin standardcurve.

Fermentation and Enzyme Recovery

F. heparinum (Payza, A. N., and Korn, E. D. (1956) Nature 177, 88-89)(ATCC 13, 125) was stored at -70° C. in a defined medium containingdimethyl sulfoxide (Me₂ SO) (Zimmermann, J. J., Oddie, K., Langer, R.,and Cooney, C. L. (1991) Appl. Biochem. Biotech. 30, 137-148). Theorganism was grown in a two liter stirred tank fermenter on heparin asthe sole carbon source in defined medium by the method of Galliher, P.M., Cooney, C. L., Langer, R. S., and Linhardt, R. J. (1981) Appl.Environ. Microbiol. 41, 360-365). From 5 liters of fermentation broth,an 80 g wet cell pellet was obtained by centrifugation for 15 min at12,000×g at 4° C. This pellet was suspended in 500 ml of 10 mM sodiumphosphate buffer at pH 7.0 and 4° C. Cell suspension (20 ml at a time)was placed into a 50-ml stainless steel cup and sonicated with coolingfor 10 min at 100 watts using a 40% pulsed mode. The disrupted cellswere centrifuged at 12,500×g for 30 min at 4° C. and the pelletdiscarded. The 500 ml of supernatant, obtained by sonification andcentrifugation, contained 16.3 mg/ml protein. Protamine free base (2.0g) was dissolved in 20 ml of 10 mM sodium phosphate buffer, pH 7.0, andadded dropwise with stirring to the 500 ml of supernatant.Centrifugation at 10,000×g, at 4° C. for 20 min, removed theprecipitated DNA and gave 510 ml of supernatant.

Purification of heparin Lyases from F. heparinum

Batch Hydroxylapatite Adsorption and Release

The 510 ml of supernatant containing 15.6 mg/ml protein, used directlywithout freezing, was divided equally into four 250 ml polypropylenecentrifuge containers and placed in an ice bath. Dry hydroxylapatite(HA) (20 g) was added to each container, gently stirred, lightlycompacted by centrifugation at 1000×g for 2 min at 4° C., and thesupernatant was decanted away from the HA matrix. The HA-bound proteinwas then resuspended in buffers having increasing concentrations ofsodium phosphate and sodium chloride and recompacted by centrifugation.The supernatants were again decanted away from the matrix and assayedfor enzyme activity and protein concentration. The buffers used to washthe HA matrix were prepared by mixing a solution of 10 mM sodiumphosphate buffer at pH 6.8, with a solution of 250 mM sodium phosphatebuffer at pH 6.8, containing 500 mM sodium chloride in ratios of 6:0,5:1, 4:2, 3:3, 2:4, and 0:6 (v/v) at 4° C. The protein supernatantsolutions were placed in dialysis tubing having a molecular weightcut-off of 14,000 and dialyzed overnight at 4° C. against 50 mM sodiumphosphate buffer at pH 7.0.

QAE-Sephadex Chromatography

Lyase activity purified by batch HA was used immediately withoutfreezing. A quaternary ammonium ethyl (QAE)-Sephadex chromatography stepwas performed at 4° C. Three batch HA-purified fractions (4:2; 3:3, and2:4), having a total volume of 1.5 liters, containing more than 89% ofthe activity toward heparin and 88% of the activity toward heparansulfate were consolidated (1.81 mg/ml protein and 1.72 units/ml towardheparin and 2.16 units/ml toward heparan sulfate) and applied directlyin equal portions to three columns (2.5×20 cm) containing 600 ml ofQAE-Sephadex. The QAE-Sephadex columns had been previously equilibratedwith 50 mM sodium phosphate buffer, pH 7.0, at 4° C. Each column wasthen washed with 1-column volume of 50 mM phosphate buffer, pH 7.0, at4° C. The fractions containing lyase activity that passed through thecolumns without interaction were collected and combined. The 2.6 litersof eluent was then concentrated to 63 ml (containing 8.23 mg/ml ofprotein) by Amicon pressure filtration at 60 psi and 4° C. using a 43 mmPM-10 membrane (10,000 molecular weight cut-off).

Hydroxylapatite HPLC

The 63 ml of QAE-Sephadex-purified and concentrated solution was dividedinto twelve 5 ml aliquots and stored at -70° C. until needed. A 5 mlsample (43 mg of protein) was removed from the freezer, allowed to thawat room temperature, and, using a 5 ml loop, injected onto a HA HPLCcolumn. The HA-HPLC column had been equilibrated with 50 mM sodiumphosphate buffer, pH 7.0. After loading the sample, the column waswashed with 50 mM sodium phosphate buffer, pH 7.0, at 0.5 ml/min, for 20min. A 60 ml linear gradient, from 50 mM sodium phosphate, pH 7.0, to 50mM sodium phosphate buffer containing 750 mM sodium chloride, pH 7.0,was used to elute the column. The elution was monitored continuously at280 nm. After the gradient was complete, the column was washed with 5.0ml of 50 mM sodium phosphate containing 1M sodium chloride, pH 7.0, toremove tightly bound proteins, and then re-equilibrated with the 50 mMsodium phosphate buffer, pH 7.0. This fractionation step was repeatedwith the 11 remaining aliquots. The fractions corresponding to heparinlyase I, heparin lyase II, and heparin lyase III from each of the 12fractionations were pooled, dialyzed against 20 volumes of 50 mM sodiumphosphate buffer, pH 7.0, for 12 h at 4° C., and concentrated at 60 psiand 4° C. using Amicon pressure filtration equipped with PM-10membranes. The three lyase preparations were each divided into 1-mlaliquots and frozen at -70° C.

Mono-S FPLC of heparin Lyases I and III

The concentrated heparin lyase I and heparin lyase III preparations,isolated from HA-HPLC, were taken from the -70° C. freezer, thawed atroom temperature, and applied to a Mono-S FPLC HR 5/5 cation-exchangecolumn equilibrated with 50 mM sodium phosphate buffer, pH 7.0. Aportion of each lyase preparation, 350 μl containing 1.75 mg of protein,was injected and the column washed at 1 ml/min for 5 min with 50 mMsodium phosphate buffer, pH 7.0, to elute non-interacting proteins. Alinear gradient from 50 mM sodium phosphate buffer, pH 7.0, to 50 mMsodium phosphate containing 500 mM sodium chloride, pH 7.0, was used andthe elution was monitored at 280 nm. The active heparin lyase I andheparin lyase III fractions were dialyzed at 4° C. against 200 mM sodiumphosphate buffer, pH 7.0, for 12 h and concentrated using AmiconPressure Filtration with a PM-10 membrane (molecular weight cut-off10,000).

Gel Permeation HPLC

The heparin lyase I and III preparation obtained from Mono-S FPLC andthe heparin lyase II preparation obtained from HA-HPLC were applied to aBio-Sil gel permeation chromatography (GPC) HPLC column (1×25 cm) thathad been equilibrated with 200 mM sodium phosphate buffer, pH 7.0. Eachlyase was injected (250 μl samples containing 800 μg of protein forheparin lyases I and III; 200 μl samples containing 1.5 mg of proteinfor heparin lyase II), eluted at a flow rate of 1 ml/min and absorbanceat 280 nm was measured. This separation was repeated 5 times for heparinlyases I-III. The active fractions were pooled together and assayed forlyase activity and protein concentration. Each heparin lyase wasdialyzed against 50 mM sodium phosphate buffer, pH 7.0, concentrated at60 psi and 4° C. using pressure filtration with 25 mm PM-10 membranes(molecular weight cut-off 10,000), and subdivided into 10 μl aliquotsand stored at - 70° C.

Characterization of the Three heparin Lyases

Assessment of Purity by Electrophoresis

Discontinuous SDS-PAGE was performed on the three heparin lyases using amodification of a procedure previously described by Laemmli, U. K.(1970) Nature 227, 680-685 (FIG. 4). The gels were fixed with 12% (w/v)trichloroacetic acid, rinsed with deionized, distilled water and stainedwith a Rapid Coomassie Stain solution, and destained.

IEF gel electrophoresis was run on pre-cast agarose gels (85×100 mm).Two electrode wicks were wetted with 1M phosphoric acid (anolyte) and 1Msodium hydroxide (catholyte). Electrophoresis was at 5 watts for 5 min,then at 10 watts for 1 h until the voltage was constant at 1200 V. Thegel was immediately fixed in 15% aqueous trichloroacetic acid, blottedand rinsed with water, dried overnight, stained by using CoomassieG-250, and destained.

Continuous acid-urea gel electrophoresis was performed in 10%polyacrylamide tube gels (Panyim, S., and Chalkley, R. (1969) Arch.Biochem. Biophys. 130, 337-346). Heparin lyase I-III samples (10 μg)were prepared in acetic acid-urea buffer containing glycerol andnaphthol red as a tracking dye. Electrophoresis was at a constantcurrent of 2.5 mA/tube gel. The proteins were run toward the cathode forapproximately 2 h, until the 100 μg of cytochrome c standard (a brownband) was at the bottom of its tube. Staining and destaining wereaccomplished as described for SDS-PAGE.

Capillary zone electrophoresis on the three heparin lyases used a DionexCapillary Electrophoresis System on a 375 μm×70-cm capillary by apreviously published method for protein analysis (Lauer, H. H., andMcManigill, D. (1986) Anal. Chem. 58, 166-170) in 20 mM CAPS containing10 mM potassium chloride, pH 11.0, at 20 kV at room temperature anddetection was by absorbance at 280 nm. Heparin lyase I-III samples (20nl), each containing 2.74, 2.07, and 2.45 mg/ml, respectively, wereanalyzed.

Reversed-phase HPLC

Reversed-phase (RP) HPLC (HP-1090 Hewitt Packard, CA) used a Vydac C₁₈column (Sasisekharan, R. (1991) Ph.D. thesis, Cloning and BiochemicalCharacterization of heparinase from Flavobacterium heparinum, HarvardUniversity). One nmol of each purified enzyme was injected onto theRP-HPLC column and eluted using a gradient from 0 to 80% acetonitrile in0.1 to 1 TFA, H₂ O for 120 min. These elution profiles were monitored at210 and 280 nm. The enzyme peaks were isolated for amino acid analysisfor composition and digestion with trypsin for peptide mapping.

Tryptic Peptide Mapping

A nanomole of each RP-HPLC-purified enzyme was denatured in 50 μl of 8Murea containing 400 mM ammonium carbonate and 5 mM dithiothreitol at 50°C. (Sasisekharan, R. (1991) Ph. D. thesis). After cooling to roomtemperature, the proteins were alkylated with 10 mM iodoacetamide for 15min in the dark. The total reaction volume was 200 μl. Trypsin (4%, w/w)was added to each lyase solution, and the proteins were digested at 37°C. for 24 h. Proteolysis was terminated by heating at 65° C. for 2 min.The peptides formed in each digest were completely soluble and wereinjected onto RP-HPLC column and were eluted using a gradient from 0 to80% acetonitrile in 120 min. The tryptic peptide maps were monitored at280 nm.

Amino Acid Compositional and N-terminal Analysis

Amino acid compositional analysis was performed at the BiopolymersLaboratories at the Massachusetts Institute of Technology on an AppliedBiosystems model 420/130 Derivatizer/Amino Acid Analyzer usingphenylisothiocyanate pre-column derivatization chemistry. Gas-phasehydrolysis of samples was performed using a Waters Pico Tag HydrolysisWorkstation. In pre-column derivatization, free amino acids are coupledwith phenylisothiocyanate to form phenylthiocarbamyl amino acids thatwere detected at 254 nm as they eluted from the reversed-phase column.Hydrolysis used 6N hydrochloric acid, 0.1% phenol at either 155° C. for1 h or 100° C. for 22 h. Hydrolysis times of 36 and 48 h were alsoexamined to ensure that the protein was being fully hydrolyzed withminimum destruction of amino acid residues N-terminal analysis was doneon 1 nmol of heparin lyase I-III.

Effect of pH on Activity

The activity pH optimum for each of the lyases was obtained by usingsuccinic acid (4.0-6.5), bis-tris propane (BTP)-HCl (6.5-9.0) and bothTris-HCl and sodium phosphate (6.0- 7.5). Heparin lyase I-III assaysolutions were made by diluting a 10-μl sample of the purified lyase(2-3 mg/ml protein concentration) with 90 μl of sodium phosphate bufferat 50 mM, pH 7.0, and placed on ice until required for assay. Theactivities of each lyase (I acting on heparin, II acting on both heparinand heparan sulfate, and III acting on heparan sulfate) were thendetermined at different pH values.

Buffer Selection for Optimum Activity

The buffer giving optimum activity for each heparin lyase was selectedby testing buffers with buffering capacity near the pH optima calculatedin the previous experiments. These buffers were: Tris-HCl sodiumphosphate, HEPES, MOPS, TES, and BTP-CHl Each buffer was prepared at 50mM, and its pH was adjusted with hydrochloric acid or sodium hydroxideto 6.9 for heparin lyase II acting on heparin, 7.15 for heparin lyase I,7.3 for heparin lyase II acting on heparan sulfate, and 7.6 for heparinlyase III. The heparin lyase assay solutions were made by dilutingenzyme in 50 mM sodium phosphate buffer adjusted to the appropriate pHas previously described. Heparin lyase activity was determined in eachbuffer. Activity was assayed both immediately after addition to eachbuffer and following incubation for 24 h at 37° C.

Affect of Divalent Metals and Added Salt on Activity

BTP-HCl buffer (50 mM) was prepared containing either 10 mM calciumchloride, 10 μM or 1 mM copper (II) chloride, 10 μM and 1 mM mercury(II) chloride, and 1 mM zinc (II) chloride. Each solution was adjustedto the optimum pH for the lyase being tested, and the activity of theheparin lyases was measured in the presence and absence of divalentmetals.

The salt concentration for optimum activity was investigated. Sodiumchloride, potassium chloride, and sodium and potassium acetate were usedto differentiate between ionic strength and specific ion affects. Addedsalt concentrations varied between 0 and 500 mM and were prepared in 50mM sodium phosphate buffer after which the pH was adjusted to eachenzyme's optimum and the heparin lyase activity was measured.

Temperature for Optimum Activity

Temperature for optimum activity was determined for the heparin lyasesat their optimum pH in sodium phosphate buffer (the heparin lyase Iassay buffer contained 100 mM sodium chloride) in 5° increments attemperatures between 15° and 55° C. The temperature was adjusted in atemperature-regulated spectrophotometer and equilibrated for 10 minbefore the assay was started.

Temperature Stability Optima

Lyase assay stock solutions were prepared in the appropriate buffer andplaced in water baths at the following temperatures: heparin lyase I at30° C. heparin lyase II at 35° C., and heparin lyase III at both 35 and40° C. Small aliquots were taken out at various time intervals (1-22 h)to measure remaining enzyme activity.

Determination of Kinetic Constants

Michaelis-Menten constants were determined using the optimizedconditions. The final absorbance value for total depolymerization wasdivided by 20 to find a value that represented 5% reaction completion.The purified lyase preparations were diluted so that 5% of totaldepolymerization would be reached only at the end of a 3-min assay. Thereaction velocities at specific molar concentrations for each lyase andtheir substrates were used for kinetic analysis using EZ-FIT hyperboliccurve-fitting program of Perella, F.W. (1988) Anal. Biochem. 174,437-447). Substrate solutions were prepared from 50 mg/ml heparin and 40mg/ml for heparan sulfate stock solutions. These constants weredetermined at 30° C. in 50 mM sodium phosphate buffer at pH 7.15containing 100 mM sodium chloride for heparin lyase I and 35° C. forheparin lyase II in 50 mM sodium phosphate buffer at pH 7.3 for heparinand pH 6.9 for heparan sulfate and at 35° C. in 50 mM sodium phosphatebuffer at pH 7.6 for heparin lyase III.

Activity of the heparin Lyases on Complex Polysaccharides

Each heparin lyase was added to a solution of complex polysaccharides (1mg/ml) under optimized assay conditions, and the reaction was monitoredat 232 nm for 30 min. The amount of purified lyase used was sufficientfor complete depolymerization of heparin or heparan sulfate substrateswithin 30 min. The initial rate of depolymerization of eachpolysaccharide was measured, the reaction was then continued for 24 h,and the final level of polysaccharide depolymerization was assessed bymeasuring the final absorbance at 232 nm and expressed as percentactivity.

Stability of the Heparin Lyases

Heparin lyase stabilities toward freeze thawing and lyophilization wereinvestigated using two excipients, bovine serum albumin (BSA) at 2 mg/mland lactose at 0.5 wt %. Each lyase was either dissolved in 50 mM sodiumphosphate buffer, 50 mM sodium phosphate buffer containing 2 mg/ml BSA,or 50 mM sodium phosphate buffer containing 0.5% lactose atconcentrations of 1-3 units/ml. These lyase solutions were then dividedinto 3 equal aliquots, and one of each was subjected to either freezethawing, lyophilization, or retained as a control in an ice bath. Theactivities of heparin lyases I-III were determined in the presence andabsence of excipients after: 1) brief storage at 4° C. 2) freezing at-70° C. and thawing; and 3) --70° C. freezing, lyophilization, andreconstituting with an equal volume of cold water.

Results

Optimized cell lysis of F. heparinum by sonication was accomplished in10 min at 100 watts using a 40% pulse mode without inactivation of theliberated enzyme. Protamine precipitation increases both the total andspecific activity by 42-fold without decreasing protein concentration,presumably by removing the polyanionic nucleic acids that maycompetitively inhibit the heparin lyases. A batch HA purification stepgreatly reduces the protein concentration and other contaminatingactivities associated with heparin/heparan sulfate metabolism, but doesnot separate the three heparin lyase activities. QAE-Sephadex is used toremove contaminating acidic proteins. HA-HPLC resolves the three lyaseactivities. A linear sodium chloride gradient is used to elute heparinlyases I-III at 330, 555, and 435 mM sodium chloride, respectively, asshown in FIG. 1. Chondroitin/dermatan sulfate lyases, also found in thisbacterium, elute from the HA-HPLC column at the end of the gradient,just behind heparin lyase II. This technique gave good recovery of totalheparin lyase activity while reducing protein concentration. Heparinlyases I and III were further purified by cation exchange FPLC, as shownin FIG. 2. Heparin lyase I is recovered with excellent retention ofactivity and a large decrease in protein concentration. The specificactivity of heparin lyase III does not improve using Mono-S FPLC, as itshowed a substantial reduction in total activity. SDS-PAGE analysis,however, revealed an improvement in the purity of heparin lyase IIIfollowing this step. Heparin lyase II was not purified by Mono-S FPLC,since it does not bind to the column. In the final purification step,heparin lyases I-III were fractionated using GPC, as shown in FIG. 3.

Following GPC each heparin lyase preparation was shown to be homogeneousby SDS-PAGE, acid-urea PAGE, IEF, capillary zone electrophoresis, andreverse phase HPLC. The molecular weights estimated by SDS-PAGE fromheparin lyases I-III were 42,800, 84,100, and 70,800, respectively.

The results obtained using this purification scheme for the threeheparin lyases are summarized in Table I. Heparin lyase I was purified3400-fold over the cell homogenate. The scheme provided on overall yieldbased on mass of 0.03%, a yield based on total activity recovery of10.8%, and had a specific activity of 130 units/mg. Heparin lyase II waspurified 5200-fold over the cell homogenate with an overall yield basedon a mass of 0.02%. This enzyme had a specific activity of 19 units/mgtoward heparin with a 1.02% total activity recovery. This enzymepreparation also had a specific activity of 36.5 units/mg toward heparansulfate, a 1.54% total activity recovery. Heparin lyase III was purified5100-fold over the cell homogenate, a yield of based on mass of 0.02%, ayield based on total activity of 2.74%, and had a specific activity of63.5 units/mg.

                  TABLE I                                                         ______________________________________                                        Purification summary of the heparin lyases                                                Protein Activity          %                                       Purification step                                                                         mg      units    Unit/mg  Activity                                ______________________________________                                        Heparin lyase I                                                               Cell homogenization                                                                       8150    66       8.12 × 10.sup.-3                           Protam Ppt. 7960    2890     3.63 × 10.sup.-1                                                                 100                                     Batch-HA    2720    2580     9.50 × 10.sup.-1                                                                 89.4                                    QAE Sepharose                                                                             519     2220     4.27     76.8                                    HA-HPLC     22.6    944      41.8     32.7                                    Mono-S FPLC 7.36    877      119      30.4                                    GPC-HPLC    2.40    313      130      10.8                                    Heparin lyase II                                                              acting on heparin                                                             Cell homogenization                                                                       8150    66       8.12 × 10.sup.-3                           Protam Ppt. 7960    2890     3.63 × 10.sup.-1                                                                 100                                     Batch-HA    2720    2580     9.50 × 10.sup.-1                                                                 89.4                                    QAE Sepharose                                                                             519     2220     4.27     76.8                                    HA-HPLC     19.6    109      5.53     3.8                                     GPC-HPLC    1.55    29.4     19       1.02                                    Heparin lyase II                                                              acting on heparan                                                             sulfate                                                                       Cell homogenization                                                                       8150    91.5     1.13 × 10.sup.-2                           Protam Ppt. 7960    3680     4.63 × 10.sup.-1                                                                 100                                     Batch-HA    2720    2580     1.19     88.0                                    QAE Sepharose                                                                             519     2220     4.11     57.8                                    HA-HPLC     19.6    275      14       7.46                                    GPC-HPLC    1.55    56.5     36.5     1.54                                    Heparin lyase III                                                             Cell homogenization                                                                       8150    91.5     1.13 × 10.sup.-2                           Protam Ppt. 7960    3860     4.63 × 10.sup.-3                                                                 100                                     Batch-HA    2720    3420     1.19     88.0                                    QAE Sepharose                                                                             519     2130     4.11     57.8                                    HA-HPLC     23.1    1010     43.6     27.4                                    Mono-S FPLC 8.41    348      41.4     9.45                                    GPC-HPLC    1.59    101      63.5     2.74                                    ______________________________________                                    

Characterization of heparin. Lyase Purity and Physical Properties Thephysical, kinetic, and stability characteristics of the three heparinlyases were investigated. Discontinuous SDS-PAGE (Laemmli, U. K. (1970))illustrated the three heparin lyases were apparently homogeneous. Themolecular weights of heparin lyase I, III were estimated at 42,800,84,100, and 70,800, respectively. Nonreducing SDS-PAGE withoutβ-mercaptoethanol revealed the same banding pattern, suggesting that nosubunits were present. IEF was used to determine the isoelectric pointsof the three heparin lyases and to assess their purity. IEF using avariety of pH gradients (pH 3-10, 7-10, and 8.5-10.5) failed to giveaccurate pI values for the three lyases as they each migrated to aposition very near the cathode. An agarose gel with a pH gradient of9-11 was then used, focusing the three proteins below the band forcytochrome c standard (pI=10.25). The pI values measured for heparinlyases I-III were 9.1-9.2, 8.9-9.1, and 9.9-10.1, respectively.Urea-acetic acid PAGE in tube gels, using the method of Panyim, S., andChalkley, R. (1969), confirmed the homogeneity of the three heparinlyases. Capillary zone electrophoresis electropherograms (Lauer, H. H.,and McManigill, D. (1986)) of each heparin lyase gave a singlesymmetrical peak. Heparin lyases I-III had migration times of 12.7,12.4, and 13.4 min, respectively.

RP-HPLC was used to desalt the three heparin lyases prior to amino acidcompositional analysis and tryptic digestion for peptide mapping(Sasisekharan, R. (1991) Ph. D. thesis). Interestingly, eachchromatogram shows a very tight doublet of peaks suggesting the presenceof isoforms, possibly due to post-translational modification. Amino acidanalysis of heparin lyase isoforms for I, II, and III were identical.The isoforms differ slightly in hydrophilicity, possibly due to somepost-translational modification such as glycosylation orphosphorylation. The major isoform of heparin lyases I-III had retentiontimes of 38.5, 44.3, and 42.7 min, respectively, in a RP-HPLC.

The major RP-HPLC peak corresponding to each heparin lyase was treatedexhaustively with trypsin to prepare peptide fragments. These peptidefragments were again analyzed using RP-HPLC. As shown in FIG. 6A and 6B,the peptide map of each lyase was distinctly different although a fewcommon peptide fragments were observed.

Amino acid analyses of the three heparin lyases are shown in Table II.The N-terminal amino acid is modified and hence cannot be detected byamino acid sequencing for all three lyases.

The amino acid composition and peptide mapping demonstrate that heparinlyases I-III are different gene products and that heparin lyases I andIII are not merely post-translationally processed from the largerheparin lyase II.

The lyases all contain a high amount of lysine that may contribute totheir high isoelectric points. Computer modeling, using the amino acidcomposition of heparin lyase I, gave a calculated isoelectric point of9.33 in agreement with the experimental values obtained by usingisoelectric focusing.

                  TABLE II                                                        ______________________________________                                        Amino Acid Composition for Heparinase I, II and III                           Amino Acid                                                                             Heparinase I                                                                              Heparinase II                                                                            Heparinass III                                ______________________________________                                        ASX      45          91         95                                            GLX      36          62         67                                            SER      24          37         38                                            GLY      30          101        50                                            HIS       6          14         13                                            ARG      13          37         35                                            THR      20          35         25                                            ALA      26          55         52                                            PRO      20          40         35                                            TYR      27          54         37                                            VAL      18          44         37                                            MET       2          15          7                                            ILE      20          31         24                                            LEU      17          53         42                                            PHE      17          35         36                                            LYS      47          47         40                                            ______________________________________                                         Assuming 727 amino acids for heparinase II (84,000 daltons), and 636 amin     acids for heparinase III (70,000 daltons). Cys and Trp not reported.     

Characterization of Optimal Catalytic Activity for the Heparin Lyases

The optimal reaction conditions for each of the three heparin lyases wasdetermined in a series of experiments. The first parameter examined wasthe pH optimum. A heparin concentration of 2.5 mg/ml for heparin lyasesI and II and a heparan sulfate concentration of 1.0 mg/ml for heparinlyases II and III were demonstrated to be saturating based on publishedvalues (14, 26) and preliminary experiments. A reaction temperature of37° C. was initially chosen as an average of values reported in theliterature (Linhardt, R. J., Turnbull, J. E., Wang, H. M., Loganathan,D., and Gallagher, J. T. (1990); Silva, M. E., Dietrich, C. P., andNader, H. B. (1976); Yang, V. C., Linhardt, R. J., Berstein, H., Cooney,C. L., and Langer, R. (1985)). The temperature was later modified afterthe optimum for each lyase was determined.

The pH optima determined were 7.15 on heparin for lyase I, 7.3 onheparin and 6.9 on heparan sulfate for lyase II, and 7.6 on heparansulfate for lyase III.

The buffer giving optimum activity for each heparin lyase was selectedusing six different buffers each adjusted to the optimum pH for theenzyme and substrate being studied. Heparin lyase I showed similarinitial reaction velocities in Tris-HCl and BTP-HC1, intermediateactivity in sodium phosphate, and reduced activity in MOPS, TES, andHEPES. After incubation in each buffer at 37° C. for 24 h, the activitywas reduced to 1-20% of its initial value. Heparin lyase I incubated inMOPS, TES, and HEPES retained the most activity. Heparin lyase IIactivity on heparin was remarkably similar in all six buffers. Whenacting on heparan sulfate, however, heparin lyase II also showed amarked reduction of activity in MOPS, TES, and HEPES. After incubationin each buffer, MOPS, TES, and HEPES were found to best protect heparinlyase II activity (30-70% retention of activity) toward both heparin andheparan sulfate. Heparin lyase III showed only slight differences inactivity in the six buffers studied. MOPS and HEPES protected heparinlyase III activity (15-30% retention of activity) following incubation.

The affect of calcium, copper (II), mercury (II), and zinc (II) ions onheparin lyase initial reaction velocities were investigated based onprior literature (Silva, M. E., Dietrich, C. P., and Nader, H. B.(1976); Hovingh, P., and Linker, A. 1970)). BPT-HCl buffer (50 mM) waschosen because of its compatibility with these ions.

The ionic strength (0-500 mM) for optimum activity was investigated foreach heparin lyase at its pH optimum in 50 mM sodium phosphate buffer.Sodium chloride, potassium chloride, sodium acetate and potassiumacetate gave comparable activities at the same ionic strength. Heparinlyase I showed increased activity in response to increased saltconcentrations, with an optimum activity at 100 mM. Heparin lyases IIand III each show a decrease in activity with increasing concentrationof added salt. At 400 mM of salt, the activity heparin lyase I-III werealmost completely inhibited.

The temperature for optimum activity was determined for the heparinlyases in 50 mM sodium phosphate buffer at their optimum pH (withheparin lyase I containing 100 mM sodium chloride) using temperaturesbetween 15 and 55° C. The temperatures for maximum activity were 35° C.for heparin lyase I, 40° C. for heparin lyase II acting on both heparinand heparan sulfate, and 45° C. for heparin lyase III. The temperaturestability optima for the heparin lyases were established to ensure thatthermal inactivation did not influence experiments aimed at determiningthe kinetic constants. Heparin lyases I and III (protein concentrationof 650 ng/ml) showed an exponential decrease in activity. Heparin lyaseI lost 80% of its activity in 5 h at 30° C. Heparin lyase III lost 80%of its activity in 3.5 h and 0.5 h at 35° C. and 40° C. respectively.Heparin lyase II (protein concentration 1-2 μg/ml) showed a much slowerdecay in activity, retaining 70% of its activity on both heparin andheparan sulfate after 25 h at 35° C. All further studies on heparinlyase I-III used 30, 35, and 35° C., respectively, to retain highactivity while maintaining enzyme stability.

The heparin lyases showed less than 0.5% activity toward chondroitinsulfate C and dermatan sulfate and no activity toward chondroitinsulfate A, D, and E. No hyaluronidase, glucuronidase activity and lessthan 0.5% sulfatase activity was observed.

The specificity of the three heparin lyases was examined using theirpolysaccharide substrates. The initial rate and the final level ofheparin and heparan sulfate depolymerization was measured. Heparin lyaseI-III acted at an average of 7, 14, and 1 sites in the heparin polymerand 5, 25, and 20 sites in the heparan sulfate polymer, respectively.Heparin lyase II acted on heparan sulfate at 1.7 times the initial rateobserved on heparin. Oligosaccharide maps, in which the oligosaccharideproducts are analyzed by strong anion-exchange HPLC and gradient PAGE(Linhardt, R. J., Turnbull, J. E., Wang, H. M., Loganathan, D., andGallagher, J. T. (1990)), were prepared for each heparin lyase acting onheparin and heparan sulfate (Lohse, D. L. (1992) Ph. D. thesis, TheHeparin lyases of Flavobacterium heparinum, The University of Iowa).These data are consistent with the specificity for heparin lyase I-IIIshown in FIG. 5.

Determination of the Michaelis-Menten Constants for the Heparin Lyases

Michaelis-Menten constants were determined using the optimum reactionconditions in experiments designed to calculate reaction velocities ateach substrate concentration where less than 10% had been consumed(Table III).

Stability of heparin Lyases

It was necessary to study conditions for the optimal storage of theheparin lyases as the literature is replete with examples of theinstability of these enzymes. In the absence of excipient, heparin lyaseI stored at 4° C., after a single freeze-thawing and afterfreeze-drying, retained 50, 45, and 25% of its activity, respectively.The addition of 2.0 mg/ml BSA enhanced storage stability, resulting ingreater than 85% retention of activity, as did the addition of 5%lactose, giving 40-80% retention of activity. Heparin lyase II retainedgreater than 75% of its activity under all storage conditions, and theaddition of BSA or lactose gave little additional stabilization. Heparinlyase III is very unstable toward freeze-thawing and lyophilization.Heparin lyase III retains most of its activity during brief storage at4° C. but lost 70-80% on freeze-thawing or freeze-drying. The presenceof BSA increases the recovered activity by 20-25% but added lactosedestabilizes heparin lyase III.

                  TABLE III                                                       ______________________________________                                        Kinetic constants of the purified heparin lyases                              Heparin                                                                       lyase  Substrate   K.sub.m(app).sup.a                                                                      V.sub.max.sup.a,b                                                                      K.sub.cat /K.sub.m c                    ______________________________________                                        I      Heparin     17.8 ± 1.50                                                                           219 ± 3.48                                                                         8.82                                    II     Heparin     57.7 ± 6.56                                                                           16.7 ± 0.555                                                                        0.405                                  II     Heparan sulfate                                                                           11.2 ± 2.18                                                                          28.6 ± 1.26                                                                         3.57                                    III    Heparan sulfate                                                                           29.4 ± 3.16                                                                           141 ± 3.88                                                                         5.59                                    ______________________________________                                         .sup.a Values of the apparent K.sub.m and V.sub.max are derived from          initial velocities obtained at eight or more concentrations (3-500 μM)     of either heparin or heparan sulfate. Protein concentrations for heparin      lyases I-III were 80, 994 and 68 ng/ml, respectively. Standard errors of      apparent K.sub.m and V.sub.max values indicate the precision of fitting       the initial rates and corresponding concentrations of heparin or heparan      sulfate to the MichaelisMenten equation as described under "Materials and     Methods.                                                                      .sup.b V.sub.max is expressed as μmol/min mg protein.                      .sup.c K.sub.cat /K.sub. m is expressed as (sμM).sup.-1.              

The pH optimum calculated for heparin lyase I was 7.15. This value washigher than the pH of 6.5 reported by Yang et al. (1985) and by Linkerand co-workers (Hovingh and Linker, (1965 and 1970)). Both groupsassayed their lyase preparations using time periods of up to 6 h wherethermal instability might become a factor. The maximum time period usedin this study was only 3 min. The pH optimum of heparin lyase II actingon heparan sulfate was 6.9. The pH optimum for heparin lyase III was7.6. Hovingh and Linker as well as Dietrich and co-workers reported thepH optimum of between 6.0 and 7.0 for this enzyme. Again, the assay timeintervals used by both groups were up to 6 h, and the thermalinstability might account for the differences between these values.

The activity of heparin lyase I is slightly reduced by 1 mM zinc andmarkedly reduced by 10 μM and 1 mM mercury and 1 mM copper. Calcium at10 mM increased activity by 30%. The activity of heparin lyase II actingon both heparin and heparan sulfate in the presence of divalent metalions showed inhibition by all of the metals tested except for 10 μMcopper. Even calcium resulted in dramatically reduced heparin lyase IIactivity. Heparin lyase III was activated (20%) by calcium, unaffectedby copper and mercury (both at 10 μM), and inhibited by zinc, mercury,and copper (all at 1 mM). In general, the addition of divalent metalions decreased the activity of the heparin lyases. Optimal activity ofheparin lyase I was observed at an ionic strength of 100 mM. Heparinlyases II and III activity decreases with increasing saltconcentrations.

Table III summarizes the apparent Michaelis-Menten constants for heparinlyases I-III acting on heparin and heparan sulfate. Apparent K_(m)values for heparin lyase I ranging from 0.3 to 42 μM and a V_(max) of19.7 μmol/min/mg protein have been reported (Rice, et al., (1989); Yang,et al., (1985); Lindhardt, (1984)). An apparent K_(m) of 5.7 μM andV_(max) of 3.57×10⁻ μmol/min for a purified heparin lyase III acting onheparin sulfate have been reported (Rice and Lindhardt, (1989)).

Heparin lyase I and II act on both heparin and heparan sulfate whileheparin lyase III acts only on heparan sulfate. All three enzymes actendolytically, however, all cleavable sites within the polymer may notbe equally susceptible (Cohen, D. M. and Linhardt, R. J. (1990)Biopolymers 30, 733-741). The primary linkages within these polymericsubstrates that are cleaved by each enzyme were deduced fromoligosaccharide mapping experiments. The specificity of pure heparinlyase I-III toward heparin and heparan sulfate were identical to thatpreviously reported for their partially purified, commercially preparedcounterparts. Oligosaccharide substrates (i.e., tetrasaccharides andhexasaccharides) having equivalent sites are poor substrates. TheV_(max) /K_(m) observed for heparin lyase I and III acting ontetrasaccharide substrates is only 0.01 to 1% of the V_(max) /K_(m)measured for the polymer substrates.

The action of heparin lyases I-III on dermatan and chondroitin sulfatesA-E was also studied. These substrates vary in position and degree ofsulfation as well as the chirality of their uronic acid. The slightactivity of these enzymes toward chondroitin sulfate C and dermatansulfate suggested that either the heparin lyases are contaminated orthat these substrates contained small amounts of heparin or heparansulfate. To distinguish between these two possibilities, the reactionwas followed for longer times. All of the activity was observedinitially, after which the substrate became stable toward repeatedchallenges with fresh enzyme. This confirmed that the small activityobserved was the result of contaminated substrate (approximately 1%heparin/heparan sulfate contamination in chondroitin sulfate C anddermatan sulfate) and not contaminated enzyme. None of the heparinlyases showed activity on hyaluronic acid. The failure of the heparinlyases to act on these other glycosaminoglycans clearly demonstratesboth their specificity for heparin/heparan sulfate and the lack ofcontaminating lyase activity. No glycuronidase activity (Warnick, C. T.,and Linker, A. (1972) Biochemistry 11, 568-572) was observed and lessthan 0.5% sulfatase activity (McLean, M. W., Bruce, J. S., Long, W. F.,and Williamson, F. B. (1954) Eur. J. Biochem. 145, 607-615) was detectedin the purified lyases.

II. Preparation of Monoclonal Antibodies to Heparinase I, Heparinase II,and Heparinase III.

Heparin lyase I was injected into mice and their B lymphocytes used toform monoclonal antibody-producing hybridomas. The specificity of themonoclonal antibodies (MAbs) for each of the three heparin lyases wasexamined.

Materials and Methods

Preparation of heparin lyases for antibody production

Heparin lyases I, II and III were isolated from Flavobacterium heparinumand purified to homogeneity as described above. Heparin lyaseconcentrations were determined using a Bio-Rad Protein Assay Kit(Richmond, Calif., U.S.A.).

Preparation of monoclonal antibodies

Six monoclonal antibodies (mAbs) were prepared. Briefly, purifiedheparin lyase I was injected into mice three times over a period of 70days. The mouse spleens were harvested and lymphocytes were isolatedfrom the splenocyte mixture. The lymphocytes were fused with mousemyeloma cells to produce hybridomas. The hybridomas were cultured andscreened for production of antibodies to heparin lyase I. Six hybridomasfound to produce mAbs to heparin lyase I were designated M-1A, M2-B7,M2-A9, M-32, M-33, and M-34. Protein concentrations of the mAb solutionswere determined using BCA Protein Assay Reagents from Pierce (Rockford,Ill., U.S.A.).

The concentrations of each monoclonal antibody is shown in Table IV.

                  TABLE IV                                                        ______________________________________                                        MAb concentrations                                                                   MAb   (mg/mL).sup.a                                                    ______________________________________                                               M-32  49                                                                      M-33  45                                                                      M-34  41                                                                      M-1A  42                                                                      M2-A9 44                                                                      M2-B7 48                                                               ______________________________________                                         MAb solution protein concentration determined by BCA protein assay            (Pierce).                                                                

Buffers for immunoassay procedures

Nitrocellulose membranes, Goat anti-Mouse IgG (H+L) HorseradishPeroxidase (HRP) Conjugate, Tris {hydroxymethyl} aminomethane (Tris),gelatin, Tween-20 and HRP Color Development Reagent(4-chloro-1-naphthol) were purchased from Bio-Rad (Richmond, Calif.,U.S.A.). Tris buffered saline (TBS) was 20 mM Tris containing 500 mMsodium chloride, pH 7.5. Blocking solution was 3.0% gelatin in TBS.Tween-20 wash solution diluted in TBS (TTBS) was 0.05% Tween-20 in TBS.Antibody buffer was 1% gelatin in TBS. HRP color development solutionwas made by mixing 60 mg HRP Color Development Reagent in 100 mLmethanol at 0° C. with 0.015° % H₂ O₂ in TBS just prior to use.

Immunoassay analysis of heparin lyases using monoclonal antibodies

Dot-blotting immunoassay techniques were conducted as recommended in theBio-Rad Immun-Blot Assay protocol (Bio-Rad, Richmond, Calif., U.S.A.).Briefly, nitrocellulose membranes were cut to 2×3 cm pieces and 1×1 cmsquares were drawn on the membranes using a soft pencil. The membraneswere soaked in TBS for 15 minutes and air dried on filter paper for 15minutes. Various concentrations of the heparin lyase (1 μL in TBS) wereplaced in the center of each square and the membrane was air dried for15 minutes, then the membrane was immersed in blocking solution for 1hour to coat the remaining hydrophobic sites. This was washed four timesin TTBS (two quick rinses, then two 5 minute agitations), then soakedovernight in a solution of mAb 0.2% (V/V) in antibody buffer, then themembranes were washed 4 times with TTBS and added to a solution of Goatanti-Mouse-HRP (0.1% in antibody buffer) for 4 hours with gentleagitation. The membranes were washed 4 times with TTBS, then twice withTBS. HRP color development solution was added to the membranes and whenthe purple bands were clearly visible, the development was stopped byplacing the membranes in distilled water. The membranes were then driedon filter paper for 15 minutes and covered with aluminum foil to protectfrom light.

Electrophoresis

Materials

Electrophoresis was performed using a Mini-Protean II electrophoresiscell from Bio-Rad (Richmond, Calif., U.S.A.). Acrylamide andN,N'-methylene bisacrylamide were from International BiotechnologiesInc. (New Haven, Conn., U.S.A.) or used as a prepared 40% acrylamidesolution that is 37.5 acrylamide:l N,N'-methylene bisacrylamide (FischerScientific, Fairlawn, N.J., U.S.A.). Tris {hydroxymethyl} aminomethane(Tris) was from Bio-Rad (Richmond, Calif., U.S.A.).N,N,N',N'-Tetramethylethylenediamine (TEMED) was from BoehringerMannheim Biochemicals (Indianapolis, Ind., U.S.A.). Ammonium persulfate(APS) and glacial acetic acid were from Mallinckrodt Inc. (Pads, Ky.,U.S.A.). Urea and glycerol were from Fisher Scientific (Fair Lawn, N.J.,U.S.A.). Sodium dodecyl sulfate (SDS) was from BDH Chemicals, Ltd.(Poole, England). Naphthol red was from Sigma Chemical Co. (St. Louis,Mo., U.S.A.). 2-β-mercaptoethanol was from EM Science (Gibbstown, N.J.,U.S.A.). Bromophenol blue was from MCB Manufacturing Chemists, Inc.(Cincinnati, Ohio, U.S.A.). Molecular Weight Standards and RapidCoomassie Stain were from Diversified Biotech (Newton Centre, Mass.,U.S.A.)

SDS-polyacrylamide gel electrophoresis (PAGE)

Heparin lyases I, II, III and Flavobacterium heparinum cell homogenatewere analyzed using SDS-PAGE as described above. Separating gels (12%acrylamide, 10% SDS) were prepared by mixing 4.35 mL distilled water,2.5 mL of 1.5 M Tris, pH 8.8 and 3.0 mL of a commercially preparedsolution of 37.5 acrylamide:1 N,N'-methylene bisacrylamide (FischerScientific, Fairlawn, N.J., U.S.A.) as described above. This solutionwas degassed under vacuum for at least 15 minutes. Next, 50 μL of APS(10%) and 5 μL of TEMED were added to the monomer solution to initiatepolymerization. The gel solution was quickly poured between two glassplates separated by 0.75 mm spacers, overlaid with distilled watersaturated gamma-butanol and allowed to polymerize at 25° C. for 60minutes.

Stacking gel was prepared by mixing 6.4 mL distilled water, 2.5 mL 0.5MTris, pH 6.8, 1.0 mL acrylamide/Bis solution (Fischer Scientific), 50 μLAPS (10%) and 10 μL TEMED. The gamma-butanol was removed from theseparating gel, the gel was rinsed with distilled water and the stackinggel solution was carefully added to the top of the separating gel. Awell-forming electrophoresis comb was inserted in the stacking gel priorto polymerization. The stacking gel was allowed to polymerize for 60minutes and the well-forming comb was removed just prior to loading ofthe samples.

Sample buffer was prepared by mixing 4.0 mL distilled water, 1.0 mL 0.5MTris, pH 6.8, 0.8 mL glycerol, 1.6 mL SDS (10%), 0.4 mL2-β-mercaptoethanol and 0.2 mL bromophenol blue (0.05% W/V). Samples andmolecular weight standard markers for electrophoresis were diluted 1:4in sample buffer and heated for 4 minutes at 100° C. just prior toloading into the wells formed earlier in the stacking gel. Runningbuffer (0.125M Tris, 1.0M glycine, 0.5% SDS, pH 8.3) was carefullyoverlaid on the stacking gel and the electrophoresis was conducted at aconstant voltage of 200 V until the bromophenol blue marker moved towithin 0.3 cm of the bottom of the gel (typically about 45 minutes).Following electrophoresis, the gels were either electro-transferred tonitrocellulose membranes or were stained with Rapid Coomassie Stain for45 minutes followed by destaining with a 7.5% methanol/5% acetic acidsolution.

Urea/Acetic Acid-PAGE

In some experiments, an urea/acetic acid-PAGE system (Panyim, S., andChalkley, R. (1969) High resolution acrylamide gel electrophoresis ofhistones. Arch. Biochem. Biophys. 130, 337-346) was used instead ofSDS-PAGE to compare the effects of SDS on the capacity of the mAbs todetect the heparin lyases in Western blots. Stock solutions used in thepreparation of the urea/acetic acid-PAGE gels were prepared as follows.A 60% acrylamide solution was prepared by dissolving 60 g acrylamide and0.4 g N,N'-methylene bisacrylamide in 1 00 mL of distilled water. A43.2% acetic acid/TEMED stock solution was prepared by mixing 43.2 mLacetic acid, 4.0 mL TEMED and 52.8 mL distilled water. APS/urea wasprepared by dissolving 5 mg APS in 25 mL of 10M urea.

The urea/acetic acid-PAGE gels were formed by mixing 4.0 mL of 60%acrylamide solution, 3.0 mL 43.2% acetic acid/TEMED and 2.0 mL distilledwater. This solution and the APS/urea solution were degassed for 15minutes. 15 mL of the APS/urea was added to the acrylamide monomersolution, mixed and carefully poured between two glass plates separatedby two 0.75 mm spacers. A well-forming electrophoresis comb was insertedinto the top portion of the gel and the gel was allowed to polymerizefor 60 minutes.

The heparin lyases were diluted 1:4 in urea/acetic acid sample buffer.This sample buffer was prepared by mixing 520 μL acetic acid, 1.0 mLglycerol, 1.0 mg naphthol red, and 6.0 g urea in distilled water thatwas brought to a final volume of 10 mL. The well-forming comb wasremoved and samples were loaded into wells and overlaid with runningbuffer (0.9M acetic acid). Electrophoresis was conducted at a constantcurrent of 20 mA for 3 hours (prefocusing of the gel) and then at 10 mAuntil the naphthol red moved to about 0.3 cm from the bottom of the gel(about 3 hours).

Electro-transfer of heparin lyases from acrylamide gels tonitrocellulose membranes

Semi-dry transblotting was conducted using a SemiPhor Transfer Unit(TE-70) from Hoefer Scientific Instruments (San Francisco, Calif.,U.S.A.). Electro-transfer of the heparin lyases from the SDS-PAGE orUrea/acetic acid-PAGE to nitrocellulose membranes was accomplished usingSemi-dry transblotting techniques as described by Al-Hakim, A., andLinhardt, R. J. (1990) Isolation and recovery of acidic oligosaccharidesfrom polyacrylamide gels by semi-dry electrotransfer. Electrophoresis11, 23-28, except that 50 mM sodium phosphate, pH 6.8 was used as thetransfer buffer. Transfer was accomplished in 40 minutes at 8 V.

Western blot detection of the heparin lyases using the monoclonalantibodies

Heparinases on the nitrocellulose membranes were detected using Westernblotting techniques exactly as described above for dot-blottingimmunoassay procedures.

Effects of SDS on detection of monoclonal antibodies

The effects of SDS and 2-β-mercaptoethanol on the immunodetection of theheparin lyases by mAbs M-32 and M-33 were examined. Dot-blottingimmunoassays of heparin lyases I and II were performed as describedearlier except that the heparin lyases were dissolved in solutionscontaining SDS and/or 2-β-mercaptoethanol in the same proportions usedin SDS-PAGE analysis prior to blotting on the nitrocellulose membrane.

Results

The reactivity of each of the six mAbs toward the three heparin lyaseswas examined. Varying amounts of each of the three heparin lyases werespotted on nitrocellulose membranes and detected using the anti-heparinlyase mAbs followed by addition of Goat anti-Mouse IGG-HRP and colordevelopment of the immune conjugates. Table V summarizes the lowestlevels of each heparin lyases that were detected by immunoassayprocedures. As seen in Table V, the mAbs have a broad range ofsensitivities toward immunodetection of the three heparin lyases. Forinstance, M2-A9 and M2-B7 can detect as little as 10 pg of heparin lyaseII, whereas M-32, M-33 and M-34 require the presence of 1 μg of heparinlyase III in order to detect that lyase.

                  TABLE V                                                         ______________________________________                                        MAb detection of heparin lyases on nitrocellulose                             membranes.sup.a                                                               MAb   Heparin lyase I                                                                           Heparin lyase II                                                                           Heparin lyase III                              ______________________________________                                        M-32   10 ng      100 ng        1 μg                                       M-33   10 ng      10 ng         1 μg                                       M-34   10 ng      10 ng         1 μg                                       M-1A  100 pg      100 pg       10 ng                                          M2-A9 100 pg      10 pg        10 ng                                          M2-B7 100 pg      10 pg        10 ng                                          ______________________________________                                         .sup.a The minimum amount of each heparin lyase detectable by each of the     six mAb using dotblotting immunodetection.                               

These data demonstrate that mAbs can be used to distinguish betweenheparin lyases I and II when the two are present together, as in aFlavobacterium heparinum cell homogenate. Specifically, M-32 can detectlevels of heparin lyase I that are ten times lower than heparin lyaseII. Conversely, M2-A9 and M2-B7 can detect levels of heparin lyase IIthat are ten times lower than heparin lyase I. M-33, M-34 and M-1Acannot be used to distinguish between heparin lyases I and II.Furthermore, all six of the mAbs are able to detect much lower levels ofheparin lyases I and II than of heparin lyase III, thus permittingdistinction between heparin lyase III and heparin lyases I or II.Distinction between heparin lyases I and II is important because bothenzymes can act on heparin and heparan sulfate and therefore are noteasily distinguished based on their substrate specificity.

Western blot analysis of the heparin lyases

The three heparin lyases and Flavobacterium heparinum cell homogenatesamples were analyzed on SDS-PAGE followed by Western blottingimmunodetection, shown in FIG. 5a. FIG. 5b contains a typical SDS-PAGEgel of the three heparin lyases stained with Coomassie Blue along withmolecular weight markers. The ability of mAbs to detect heparin lyaseswas examined by running the three heparin lyases and Flavobacteriumheparinum cell homogenate through six SDS-PAGE gels followed by Westernblotting immunodetection of the gel contents. Heparin lyase I (18 ng),heparin lyase II (570 ng), heparin lyase III (1.63 μg) and cellhomogenate (87 ng) were loaded on each gel. The developing time used fordetection on the nitrocellulose membrane containing M-34, M1-A, M2-A9and M2-B7 were 20, 10, 15 and 40 minutes, respectively. Four of the mAbs(M-34, M-1A, M2-A9 and M2-B7) were able to detect purified heparinlyases I, II and III as well as heparin lyases present in theFlavobacterium heparinum cell homogenate. Two mAbs (M-32 and M-33) werenot able to detect either the purified heparin lyases or cellularproteins in the Western blots.

The reagent in the SDS-PAGE system that was responsible for destroyingthe ability of M-32 and M-33 to immunodetect the heparin lyases wasdetermined. Dot-blotting immunoassays of the heparin lyases using M-32and M-33 were used to evaluate each component in the SDS-PAGE system.Heparin lyases I and II, in the presence or absence of SDS and/or2-β-mercaptoethanol, were blotted on nitrocellulose membranes andexamined using dot-blotting immunoassay techniques. The mAbs were unableto detect the lyases when SDS was present, demonstrating that SDS wasresponsible for the reduction of sensitivity of these two MAbs duringthe Western blotting procedures. This experiment suggests that M-32 andM-33 must be recognizing an epitope on the lyases that requiressecondary conformation such as a folded structure present in all threeheparin lyases that is destroyed by SDS denaturation.

To further demonstrate that the SDS was responsible for the diminishedreactivity of M-32 and M-33 toward the heparin lyases, the three heparinlyases and Flavobacterium heparinum cell homogenate were analyzed usingthe urea/acetic acid-PAGE followed by Western blotting immunodetectionwith M-32 and M-33 to detect the lyases in this system. The sensitivityof detection was markedly reduced. Heparin lyase I (2.7 μg), heparinlyase II (3.4 μg), heparin lyase III (4.7 μg) and cell homogenate (7.7μg) were detectable. Thus, SDS is the agent primarily responsible forthe reduced reactivity of MAbs M-32 and M-33 toward the heparin lyases.All six MAbs are able to detect all three heparin lyases, in either thepurified or the native form, when analyzed using PAGE followed byWestern blotting immunodetection.

It was expected that at least one of the six MAbs would specificallydetect a single heparin lyase, enabling the detection of that lyase in acomplex mixture of heparin lyases such as a cell homogenate. Thedot-blotting and Western analyses revealed that all of the mAbs are ableto detect all three lyases. This observation suggests that these threeheparin lyases are remarkably similar in structure since they share sixcommon epitopes. Peptide mapping of these three enzyme demonstrates anumber of common peptide fragments and suggests that these may belocated at the highly immunogenic regions within the three heparinlyases. The sensitivities of individual mAbs toward each of the lyasesin the dot-blotting analyses varied greatly, thus offering the potentialto use the dot-blotting analysis to distinguish between the threelyases.

Use of PAGE (SDS or urea/acetic acid) required much more protein thandot-blotting procedures and the sensitivities of the mAbs toward each ofthe lyases were different than those seen in the dot-blotting analyses,probably due to alterations of secondary structure during the PAGE andtransfer steps. Thus, detection of heparin lyases using mAbs is mostefficiently conducted by use of dot-blotting techniques as describedhere. Furthermore, all six MAbs were able to detect all three lyasesthat were present in Flavobacterium heparinum cell homogenate, thusoffering the potential that these mAbs could be used to rapidlydemonstrate the presence of heparin lyases in cell homogenate. To bebeneficial in lyase purification, these MAbs must first be immobilizedand their binding avidity to the heparin lyases assessed. Methods andmaterials for immobilization of antibodies are commercially availableand known to those skilled in the art.

In summary, the results described here demonstrate that mAbs can be usedto detect heparin lyases I, II and III in either their purified state orwhen present together in a solution of homogenized Flavobacterial cells.These mAbs can also be used in dot-blotting analyses of the lyases todistinguish between the three lyases based on their differentsensitivity for each of the three lyases.

Modifications and variations of the purified heparinases, method ofpurification and monoclonal antibodies thereto will be obvious to thoseskilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe appended claims.

We claim:
 1. An isolated heparinase II from Flavobacterium heparinumfree of lyase activity other than heparinase II activity, having amolecular weight of 84,100, cleaving heparin and heparan sulfate andhaving a pH optimum of 8.9-9.1.
 2. An isolated heparinase III fromFlavobacterium heparinum free of lyase activity other than heparinaseIII activity, having a molecular weight of 70,800, cleaving heparansulfate, and having a pH optimum of 9.9-10.1.
 3. The heparinase III ofclaim 2 which does not cleave heparin sulfate.
 4. The heparinase III ofclaim 2 stabilized with albumin.
 5. A method for purifying heparinase I,II, and III from a biologically pure culture of Flavobacterium heparinumcomprising the steps oflysing Flavobacterium heparinum cells in abiologically pure culture of Flavobacterium heparinum, removing celldebri and nucleic acids from the cell lysate, absorption of heparinaseI, II, and III to hydroxyapatite, absorption of non-heparinase I, II,and III proteins to QAE-resin, recovery of the heparinase I, II, and IIInot bound to the QAE-resin, separation of heparinase I, II, and III byHPLC on a hydroxylapatite column, recovery of the heparinase I, II, andIII separated on the hydroxylapatite column, purification of theseparated heparinases by cation exchange FPLC, recovery of theheparinase I, II, and III separated by cation exchange FPLC,purification of the separated heparinases by gel permeation HPLC, andrecovering the heparinases I, II, and III separated by gel permeationHPLC.
 6. The method of claim 5 wherein the nucleic acids are removed byprecipitation with protamine.
 7. The method of claim 6 wherein theheparinases are separated on the hydroxylapatite column by elution witha salt gradient.
 8. The method of claim 6 wherein the heparinases areeluted from the cation exchange column by a gradient of increasing saltconcentration.